Multi-Potent Adult Progenitor Cells from Swine Bone Marrow.

Abstract Here, we show that Multi-potent Adult Progenitor Cells (MAPCs) can be derived from adult and fetal swine bone marrow. Swine MAPC (swMAPC) contain initially multiple different progenitors and mature cells; however, cultures are homogenous by 50 population doublings and can grow past 150 population doublings. SwMAPCs are CD44, CD45, and MHC-I, II negative, express Oct3a/4 at levels close to those seen in embryonic stem cells, and have telomerase activity leading to no telomere shortening with expansion. We also have very solid evidence that swMAPCs differentiate into most mesoderm, neuroectoderm, and endoderm lineages as demonstrated by a significant up-regulation of transcription factors and other lineage specific proteins in a time dependent fashion similar to development, measured by Q-RT-PCR as well as immunohistology. In addition, swMAPCs induced to the endothelial lineage form vascular tubes, to the hepatic lineage produce albumin and urea, and to the smooth muscle lineage display significant calcium flux in response to smooth muscle agonists. In addition, we are investigating the swMAPC differentiation into cardiomyocytes. Preliminary data indicates swMAPCs express TBX5, Nkx2.5, and GATA6 by day 7 at significant levels. As we have seen for rodent MAPCs, when swMAPCs are allowed to grow at high density, Oct3a/4 levels drop to undetectable ranges, the cells take on a mesenchymal stem cell (MSC) phenotype, and the differentiation potential is lost. When replated under low density conditions, oct3a/4 expression or differentiation capacity can not be re-induced, and cells remain MSC like. Therefore, we suggest that Oct3a/4 is not an in vitro culture phenomena but is already present in swMAPCs and cannot be recovered once lost with standard culturing techniques.

Download Full-text

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair

Scientific Reports ◽

10.1038/s41598-019-53818-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Tao-Cheng Wu ◽

Jia-Shiong Chen ◽

Chao-Hung Wang ◽

Po-Hsun Huang ◽

Feng-Yen Lin ◽

...

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Progenitor Cells ◽

Heme Oxygenase ◽

Neointimal Hyperplasia ◽

Vascular Repair ◽

Vascular Progenitor Cells

AbstractVascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

Download Full-text

Bloodstream Progenitor-Cell Traffic In Primary Myelofibrosis Reveals Cell Subsets Showing Some Features Of Very-Small Embryonnic-Like Stem Cells (VSELs), Along With Endothelial (PEC), Mesenchymal (MPC) and Hematopoietic (HPC) Progenitor Cells

Blood ◽

10.1182/blood.v122.21.5265.5265 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5265-5265 ◽

Cited By ~ 1

Author(s):

Mario Ojeda-Uribe ◽

Hanna Sovalat ◽

Laura Jung ◽

Christophe Desterke ◽

Sylvie Thiebault ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hot Spot ◽

Embryonic Stem ◽

Primary Myelofibrosis ◽

Rt Pcr ◽

In Vitro Development ◽

Oct4 Expression ◽

Vitro Development

Abstract Introduction Primary myelofibrosis (PMF) is accompanied by an increase in the bloodstream circulation of some adult progenitor cells. Extramedullary hematopoiesis observed in this setting might remind some features related to foetal hematopoiesis. Material and methods We looked for evidence in favour of this hypothesis in blood samples of a small cohort of untreated patients with PMF (4 pre-fibrotic (PF) and 4 fibrotic (F), defined according to the WHO and Thiele's histopathology score (Blood, 2011)). Patient baseline characteristics are shown below. We performed a) flow-cytometric analysis for cell subsets related to VSEL, PEC, MPC, HPC; b) RT-PCR for embryonic transcriptional factors NANOG, OCT4, SOX2, LIN28 from MNC fraction (positive control hES, negative control CPRE2 c) in-vitro development of embryonic stem like cells (ESlC) under specific culture conditions. In addition we looked for SRSF2 mutations in order to better characterize PMF stages. Results As expected we detected high numbers of circulating CD34+ cells (HPC) (mean 233083±307148/ml (range 4600-783000), with similar numbers in PF- (231125±289553/ml) and F-PMF (235040 ±369156/ml). We were able to detect small numbers of the following cell subsets related to VSEL (size 2-4m) (Fig 1) Lin-/CD45-/CD34+ (mean 124±239/ml), Lin-/CD45-/CD133+ (mean 1178±971/ml), Lin-/CD45-/CXCR4+ (mean 1572±1622/ml). Lin-/CD45-CD34+AC133+CXCR4+ cells were detected in 6 of 8 patients (mean 186±375/ml) with F-PMF patients showing higher numbers (279±416/ml) than PF-PMF (63±71/ml). NANOG and OCT4 expression was detected by RT-PCR in all the patients tested. Mean OCT4 expression was about 50% the level of hES, but F-PMF showed higher levels. NANOG expression was similar to that of hES, whereas Sox2 and Lin28 were not expressed in most patients. We failed to observe the in-vitro development of ESlC in the 2 tested patients. PEC (Lin-/CD45-CD34+AC133+KDR+) were detected in all the PF-PMF (185±332/ml) and in 1 of 4 F-PMF (mean 9±18/ml). MPC (Lin-/CD45-CD90+CD105+) were detected in higher numbers in PF-PMF (mean 413±528/ml) than in F-PMF (mean 157±216/ml). We were not able to detect mutations in the hot spot of SRSF2 (codons 93,94,95). Conclusions Small numbers of cell subsets displaying morphologic and immunophenotypic features of VSELs were detected in PMF patients. However, we are not able to define these as fully specific VSELs according to previous works that defined them (Kucia, Leukemia 2006). Interestingly Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in higher numbers in F-PMF, supporting in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the foetal period. This recruitment also involves HPC. Moreover although all patients expressed OCT4 and NANOG, OCT4 expression was higher in F-PMF. As expected PEC circulate in higher numbers in PF-PMF compared to F-PMF. Interestingly both F-PMF and PF-PMF were associated to the circulation of significant numbers of MPC but higher numbers observed in PF-MFP might be interpreted either as a necessary recruitment to establish extra-medullary stroma or due to the exit from bone marrow of highly proliferative MPC. Whether all these different circulating progenitor cells, are clonally or not clonally related to the PMF pathogenesis or to unspecific mobilisation secondary to bone marrow microenvironment injury cannot be determined from these preliminary results. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Age-Related Differences Between Unrestricted Somatic Stem Cells from Cord Blood and Bone Marrow Derived Mesenchymal Stroma Cells.

Blood ◽

10.1182/blood.v112.11.1335.1335 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1335-1335 ◽

Cited By ~ 1

Author(s):

Amelie Pia Houben ◽

Anja Buchheiser ◽

Murat Aktas ◽

Johannes Fischer ◽

Peter Wernet ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cord Blood ◽

Telomerase Activity ◽

Wnt Signalling ◽

Human Cord Blood ◽

Differentiation Potential ◽

Age Related ◽

Unrestricted Somatic Stem Cells

Abstract During the past years haematopoietic stem cells from unrelated umbilical cord blood have been increasingly used for treatment of leukaemia and genetic diseases. Human cord blood also contains a non-haematopoietic, adherently growing, CD45 negative, Oct4, nanog and Sox2 negative cell population with intrinsic multipotent differentiation potential, described as Unrestricted Somatic Stem Cells (USSC) (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Liedtke et al. 2007, Liedtke et al.2008, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). USSC can be cultivated GMP-grade reaching 1×109 cells in Passage 4–5 (correlating to 26,5 – 28 cumulative population doublings) without losing multipotency. To better understand and characterise USSC, we carried out in vitro studies concerning age-related changes in USSC-lines from human cord blood and in mesenchymal stroma cells derived from human bone marrow (BM-MSC). Telomerase activity and telomeres are involved in cell proliferation as well as the regulation of cell senescence (Lansdorp 2008). In this study 7 USSC-lines and 9 BM-MSC were analyzed for proliferation and senescence at different population doublings (PD). In vitro USSC have a higher proliferation capacity and accordingly a comparably more extended lifespan than BM-MSC. They undergo about 35 to 45 CPD whereas mean level of CPD from BM-MSC reaches 25. Telomere length of 12 USSC, 5 BM-MSC and 5 clonal populations of USSC were calculated after several PD and telomerase activity was measured. Mean terminal restriction fragment’s (TRF’s) length calculated from USSC after 31 CPD averages 9.7 kbp whereas mean telomere length from BM-MSC decreases already after 20 CPD to 7.9 kbp. After 47 CPD in clonal USSC populations mean TRF’s length is 6.1 kbp. Telomerase activity was analysed with RT-PCR and real time PCR. In contrast to previous publications telomerase activity was detected neither in BM-MSC (Parsch et al.) 2003 nor in USSCs (Manca et al. 2008). The percentage of senescent cells after the same number of CPD is significantly higher in BM-MSC than in USSC. About 80% of senescence was observed in BM-MSC but only 10% senescence in USSCs after 26 CPD. Wnt signalling is mandatory for self-renewal, cell proliferation and differentiation of haematopoietic stem cells (Reya et al. 2003). In primitive MSC populations Wnt signalling regulates mesenchymal lineage specification (Etheridge et al. 2004). For analysing the role of Wnt pathway in USSC development quantitative PCR Arrays have been carried out profiling the expression of 84 genes related to Wnt-mediated signal transduction. 8 different USSC populations have been analysed. In all USSCs factors essential for canonical and also non-canonical Wnt-signalling are present. Poor proliferating USSC with high adipogenic differentiation potential show a stronger expression of Wnt-signalling inhibitors like SFRP1, DKK1 and CXXC4. Based on age-related characteristics, USSC from cord blood are a much better source for regeneration compared to their adult MSC counterpart from bone marrow.

Download Full-text

Cell Sources for Human In vitro Bone Models

Current Osteoporosis Reports ◽

10.1007/s11914-020-00648-6 ◽

2021 ◽

Author(s):

Sana Ansari ◽

Keita Ito ◽

Sandra Hofmann

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Cell Types ◽

Medium Composition ◽

Differentiation Potential ◽

Osteoclast Progenitor ◽

Proliferation And Differentiation ◽

Cell Sources

Abstract Purpose of Review One aim in bone tissue engineering is to develop human cell-based, 3D in vitro bone models to study bone physiology and pathology. Due to the heterogeneity of cells among patients, patient’s own cells are needed to be obtained, ideally, from one single cell source. This review attempts to identify the appropriate cell sources for development of such models. Recent Findings Bone marrow and peripheral blood are considered as suitable sources for extraction of osteoblast/osteocyte and osteoclast progenitor cells. Recent studies on these cell sources have shown no significant differences between isolated progenitor cells. However, various parameters such as medium composition affect the cell’s proliferation and differentiation potential which could make the peripheral blood-derived stem cells superior to the ones from bone marrow. Summary Peripheral blood can be considered a suitable source for osteoblast/osteocyte and osteoclast progenitor cells, being less invasive for the patient. However, more investigations are needed focusing on extraction and differentiation of both cell types from the same donor sample of peripheral blood.

Download Full-text

Enrichment of blood from embryonic stem cells in vitro

Reproduction Fertility and Development ◽

10.1071/rd98104 ◽

1998 ◽

Vol 10 (8) ◽

pp. 563 ◽

Cited By ~ 5

Author(s):

Andrew C. Perkins

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Embryonic Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Leukaemia Inhibitory Factor ◽

Chimeric Mouse ◽

Differentiation Potential

Murine embryonic stem (ES) cells are pluripotent. When injected into blastocysts they can give rise to every cell type of a derived chimeric mouse including germ cells. Embryonic stem cells also possess remarkable in vitro differentiation potential. When removed from stromal support and leukaemia inhibitory factor (LIF), ES cells differentiate into structures known as embryoid bodies (EBs), in which all three germ layers develop and interact. As ES cells from humans become available there is increasing interest in the potential for EBs to provide an unlimited supply of stem cells for somatic transplantation therapies. Realisation of this potential will require greater understanding of the molecular determinants of cell fate within EBs. Also, culture techniques for selective expansion of cell lineages of interest will reduce the risks associated with transplantation of EB-derived cells. In this paper the kinetics of expression of mRNA and protein for early mesoderm markers within EBs is reported. In addition, a three-step culture system incorporating co-cultivation on the bone marrow derived stromal cell line, MC3T3-G2/PA6 (PA6), is explored as a way to select for haematopoietic progenitor cells (HPCs) and against undifferentiated ES cells. A system like this could enhance purification of haematopoietic stem cells (HSCs) from ES cells for bone marrow transplantation.

Download Full-text

The human arthritic hip joint is a source of mesenchymal progenitor cells (MPCs) with extensive multipotent differentiation potential

10.21203/rs.3.rs-17537/v1 ◽

2020 ◽

Author(s):

Mike Wagenbrenner ◽

Tizian Heinz ◽

Konstantin Horas ◽

Axel Jakuscheit ◽

Joerg Arnholdt ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hip Joint ◽

Collagen Type ◽

Hyaline Cartilage ◽

Joint Capsule ◽

Mesenchymal Progenitor Cells ◽

Differentiation Potential ◽

Monolayer Cell

Abstract Background: While multiple in vitro studies examined mesenchymal progenitor cells (MPCs) derived from bone marrow or hyaline cartilage, there is little to no data about the presence of MPCs in the joint capsule or the ligamentum capitis femoris (LCF) of the hip joint. Therefore, this in vitro study examined the presence and compared the differentiation potential of MPCs isolated from the bone marrow, arthritic hyaline cartilage, the LCF and full-thickness samples of the anterior joint capsule of the hip joint. Methods: MPCs were isolated and multiplied in adherent monolayer cell culture. Osteogenesis and adipogenesis was induced in monolayer cell cultures for 21 days using a differentiation medium containing specific growth factors, while chondrogenesis in the presence of TGF-ß1 was performed using pellet-culture for 27 days. Control cultures were maintained for comparison over the same duration of time. The differentiation process was analyzed using histological and immunohistochemical stainings as well as semiquantitative RT-PCR for measuring the mean expression levels of tissue-specific genes.Results: This in vitro research showed that the isolated cells from all four donor tissues grew plastic adherent and showed similar adipogenic and osteogenic differentiation capacity as proven by the histological detection of lipid droplets or deposits of extracellular calcium and collagen type I. After 27 days of chondrogenesis proteoglycans accumulated in the differentiated MPC-pellets from all donor tissues. Immunohistochemical staining revealed vast amounts of collagen type II in all differentiated MPC-pellets, except for those from the LCF. Interestingly all differentiated MPCs still showed a clear increase in mean expression of adipogenic, osteogenic and chondrogenic marker genes. In addition the examination of an exemplary donor sample revealed that cells from all four donor tissues were clearly positive for the surface markers CD44, CD73, CD90 and CD105 by flow cytometric analysis.Conclusions: This study proved the presence of MPCs in all four examined donor tissues of the hip joint. No significant differences were observed during osteogenic or adipogenic differentiation depending on the source of MPCs used. Further research is necessary to fully determine the chondrogenic differentiation potential of MPCs isolated from the LCF and capsule tissue of the hip joint.

Download Full-text

Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00393.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. E259-E266 ◽

Cited By ~ 80

Author(s):

Andreas Lechner ◽

Joel F. Habener

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Es Cells ◽

Embryonic Stem ◽

Pancreatic Islet Transplantation ◽

Β Cells ◽

Β Cell ◽

Adult Islet

In view of the recent success in pancreatic islet transplantation, interest in treating diabetes by the delivery of insulin-producing β-cells has been renewed. Because differentiated pancreatic β-cells cannot be expanded significantly in vitro, β-cell stem or progenitor cells are seen as a potential source for the preparation of transplantable insulin-producing tissue. In addition to embryonic stem (ES) cells, several potential adult islet/β-cell progenitors, derived from pancreas, liver, and bone marrow, are being studied. To date, none of the candidate cells has been fully characterized or is clinically applicable, but pancreatic physiology makes the existence of one or more types of adult islet stem cells very likely. It also seems possible that pluripotential stem cells, derived from the bone marrow, contribute to adult islet neogenesis. In future studies, more stringent criteria should be met to clonally define adult islet/β-cell progenitor cells. If this can be achieved, the utilization of these cells for the generation of insulin-producing β-cells in vitro seems to be feasible in the near future.

Download Full-text

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

Current Stem Cell Research & Therapy ◽

10.2174/1574888x14666190123161447 ◽

2019 ◽

Vol 14 (4) ◽

pp. 305-319 ◽

Cited By ~ 4

Author(s):

Marietta Herrmann ◽

Franz Jakob

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Progenitor Cell ◽

Adult Stem Cells ◽

Current Knowledge ◽

Cell Populations ◽

Surface Markers ◽

Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

Download Full-text

Mini Review; Differentiation of Human Pluripotent Stem Cells into Oocytes

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200116100121 ◽

2020 ◽

Vol 15 (4) ◽

pp. 301-307 ◽

Cited By ~ 3

Author(s):

Gaifang Wang ◽

Maryam Farzaneh

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Human Pluripotent Stem Cells ◽

Human Oocyte ◽

Differentiation Potential ◽

Cell Lineages ◽

Cell Based Therapy ◽

Induced Pluripotent

Primary Ovarian Insufficiency (POI) is one of the main diseases causing female infertility that occurs in about 1% of women between 30-40 years of age. There are few effective methods for the treatment of women with POI. In the past few years, stem cell-based therapy as one of the most highly investigated new therapies has emerged as a promising strategy for the treatment of POI. Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) as a type of pluripotent stem cells are the most powerful candidate for the treatment of POI. Human-induced Pluripotent Stem Cells (hiPSCs) are derived from adult somatic cells by the treatment with exogenous defined factors to create an embryonic-like pluripotent state. Both hiPSCs and hESCs can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. After ovarian stimulation, the number of available oocytes is limited and the yield of total oocytes with high quality is low. Therefore, a robust and reproducible in-vitro culture system that supports the differentiation of human oocytes from PSCs is necessary. Very few studies have focused on the derivation of oocyte-like cells from hiPSCs and the details of hPSCs differentiation into oocytes have not been fully investigated. Therefore, in this review, we focus on the differentiation potential of hPSCs into human oocyte-like cells.

Download Full-text